WO2008014198A1 - Lipoxygenase enzyme assay - Google Patents
Lipoxygenase enzyme assay Download PDFInfo
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- WO2008014198A1 WO2008014198A1 PCT/US2007/074075 US2007074075W WO2008014198A1 WO 2008014198 A1 WO2008014198 A1 WO 2008014198A1 US 2007074075 W US2007074075 W US 2007074075W WO 2008014198 A1 WO2008014198 A1 WO 2008014198A1
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- Prior art keywords
- lipoxygenase
- assay
- enzyme
- lipoxygenase enzyme
- reagent
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Links
- 102000003820 Lipoxygenases Human genes 0.000 title claims abstract description 35
- 108090000128 Lipoxygenases Proteins 0.000 title claims abstract description 35
- 238000001952 enzyme assay Methods 0.000 title description 2
- 238000003556 assay Methods 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 17
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 16
- 150000001875 compounds Chemical class 0.000 claims abstract description 15
- 239000000758 substrate Substances 0.000 claims abstract description 13
- 238000012360 testing method Methods 0.000 claims abstract description 10
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000001301 oxygen Substances 0.000 claims abstract description 9
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 9
- 239000003112 inhibitor Substances 0.000 claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims abstract description 7
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 7
- 230000005764 inhibitory process Effects 0.000 claims abstract description 7
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 7
- 239000000194 fatty acid Substances 0.000 claims description 14
- 108010029942 microperoxidase Proteins 0.000 claims description 10
- ZAIPMKNFIOOWCQ-UEKVPHQBSA-N cephalexin Chemical group C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)C)C(O)=O)=CC=CC=C1 ZAIPMKNFIOOWCQ-UEKVPHQBSA-N 0.000 claims description 7
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 7
- 229930195729 fatty acid Natural products 0.000 claims description 7
- 150000004665 fatty acids Chemical class 0.000 claims description 7
- 102000009515 Arachidonate 15-Lipoxygenase Human genes 0.000 claims description 3
- 108010048907 Arachidonate 15-lipoxygenase Proteins 0.000 claims description 3
- 230000005284 excitation Effects 0.000 claims description 3
- 102000011730 Arachidonate 12-Lipoxygenase Human genes 0.000 claims description 2
- 108010076676 Arachidonate 12-lipoxygenase Proteins 0.000 claims description 2
- 102000001381 Arachidonate 5-Lipoxygenase Human genes 0.000 claims description 2
- 108010093579 Arachidonate 5-lipoxygenase Proteins 0.000 claims description 2
- 235000021588 free fatty acids Nutrition 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000013641 positive control Substances 0.000 claims description 2
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 8
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 5
- 239000003054 catalyst Substances 0.000 description 5
- PKYCWFICOKSIHZ-UHFFFAOYSA-N 1-(3,7-dihydroxyphenoxazin-10-yl)ethanone Chemical compound OC1=CC=C2N(C(=O)C)C3=CC=C(O)C=C3OC2=C1 PKYCWFICOKSIHZ-UHFFFAOYSA-N 0.000 description 4
- 229940114079 arachidonic acid Drugs 0.000 description 4
- 235000021342 arachidonic acid Nutrition 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- HSSLDCABUXLXKM-UHFFFAOYSA-N resorufin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3N=C21 HSSLDCABUXLXKM-UHFFFAOYSA-N 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 3
- 238000002820 assay format Methods 0.000 description 3
- 238000007398 colorimetric assay Methods 0.000 description 3
- 125000001867 hydroperoxy group Chemical group [*]OO[H] 0.000 description 3
- 235000020778 linoleic acid Nutrition 0.000 description 3
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000001506 fluorescence spectroscopy Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000013537 high throughput screening Methods 0.000 description 2
- 238000006479 redox reaction Methods 0.000 description 2
- OXSANYRLJHSQEP-UHFFFAOYSA-L 4-aminophthalate Chemical compound NC1=CC=C(C([O-])=O)C(C([O-])=O)=C1 OXSANYRLJHSQEP-UHFFFAOYSA-L 0.000 description 1
- HUDPLKWXRLNSPC-UHFFFAOYSA-N 4-aminophthalhydrazide Chemical compound O=C1NNC(=O)C=2C1=CC(N)=CC=2 HUDPLKWXRLNSPC-UHFFFAOYSA-N 0.000 description 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 102000007330 LDL Lipoproteins Human genes 0.000 description 1
- 108010007622 LDL Lipoproteins Proteins 0.000 description 1
- 239000000867 Lipoxygenase Inhibitor Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010036302 hemoglobin AS Proteins 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 230000036515 potency Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- ORZHVTYKPFFVMG-UHFFFAOYSA-N xylenol orange Chemical compound OC(=O)CN(CC(O)=O)CC1=C(O)C(C)=CC(C2(C3=CC=CC=C3S(=O)(=O)O2)C=2C=C(CN(CC(O)=O)CC(O)=O)C(O)=C(C)C=2)=C1 ORZHVTYKPFFVMG-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- TECHNICAL FIELD This invention relates to methods and kits for lipoxygenase enzymes which catalyze the oxygen-dependent oxidation of fatty acid substrates (linoleic acid and arachidonic acid are common examples) to form hydroperoxy-fatty acid products.
- the methods and kits are useful for detecting inhibitors of such enzymes.
- Lipoxygenase enzymes catalyze the oxygen-dependent oxidation of fatty acid substrates (linoleic acid and arachidonic acid are common examples) to form hydroperoxy-fatty acid products. Enzymes have been purified from diverse organisms that display a broad range of substrate specificity and product specificity (i.e. the site of oxidiation within the fatty acid).
- Another assay that has been used is to determine the concentration of hydroperoxy (or the chemically-reduced hydroxy-derivatives) fatty acids by separation from the substrate on a high-performance liquid chromatography (HPLC) system (for example, Yamamoto et al. (1990) Methods in Enzymology, 186, 371-380).
- HPLC high-performance liquid chromatography
- the color- forming reagent is added and the color is measured on a spectrophotometer.
- One assay used a xylenol orange :iron(II) complex (Waslidge et al. (1995) Anal. Biochemistry, 231, 354-358) and the second assay used hemoglobin as the catalyst) and N-benzoyl leucomethylene (Auerbach et al. (1992) Anal. Biochemistry, 201, 375-380) as the colorimetric reagent.
- These assays offer improved sensitivity over the direct spectrophotometric assay ( ⁇ 10-fold) and improved throughput when compared to the HPLC method.
- the present inventors have designed an assay format to enable the identification of inhibitors of lipoxygenase enzymes. This assay represents a significant advantage over previous assay formats as the sensitivity and uniqueness of the signal render the format more amenable to high-throughput screening.
- Figure 1 Schematic depiction of microperoxidase catalyzing a redox reaction between the hydroperoxy-fatty acid product and the Amplex UltraRed® to generate the highly fluorescent product resorufin. The amount of resorufin is then determined using fluorescence spectroscopy.
- Figure 2 Schematic depiction of the fluorometric lipoxygenase assay that would be used to characterize the activity of a 15 -lipoxygenase.
- Figures 3-5 Inhibition of x-lipoxygenase by representative compounds of varying potencies.
- the y-axis is Percent of Control and the x-axis units are in microM.
- the present invention provides a new assay for lipoxygenase which is an improvement from the historical assays described above.
- the lipoxygenase has been incubated with the fatty acid substrate (linoleic acid or arachidonic acid) and oxygen, microperoxidase (a catalyst) and Amplex UltraRed® are added.
- the microperoxidase catalyzes a redox reaction between the hydroperoxy-fatty acid product and the Amplex UltraRed® to generate the highly fluorescent product resorufin.
- the amount of resorufin is then determined using fluorescence spectroscopy (excitation at 530 nm and emission at 580 nm). See figure 1.
- This assay improves the sensitivity ⁇ 10-fold over that observed in the colorimetric assays and generates a fluorescent signal that is both stable and free from compound interference as very few compounds fluoresce in this range.
- kits for determining the amount of lipoxygenase enzyme inhibition by a test compound comprising: a lipoxygenase enzyme; a lipoxygenase enzyme substrate; oxygen; a peroxidase and a fluorometric reagent.
- the above kit can further contain a positive control that comprises a mock test compound.
- Said mock test compound having no or negligible lipoxygenase enzyme inhibition.
- the Enzymes have been purified from diverse organisms that display a broad range of substrate specificity and product specificity.
- the assay as it is routinely performed is summarized in the scheme from the example section below but alterations apparent to those of ordinary skill in the art can be made. For instance, the incubation time or temperature can be adjusted but it is ideal to adjust them such that the enzyme activity is within the linear response range.
- the assay has been performed at various scales (cuvet, 96 or 384 well) and is expected to work at any scale required within any desired reaction vessel (e.g. polypropylene micro-plate or polystyrene cuvet).
- Any substrate of the lipoxygenase enzyme can be used; this could include, but is not limited to, free fatty acids or esterified fatty acids of varying composition (e.g. arachidonic acid, linoleyl- phosphatidyl choline, low-density lipoprotein, etc.).
- Amplex UltraRed® is the preferred fluorometric reagent in this protocol
- Amplex Red® or any reagent that results in the production of a fluorescent molecule with similar fluorescence can also be used.
- microperoxidase may be substituted with any peroxidase that catalyzes the reaction between the hydroperoxide product and the fluorometric reagent (i.e. Amplex UltraRed® in the preferred embodiment).
- the solutions used for the lipoxygenase reaction and the microperoxidase reaction may also be modified from the specified conditions so long as activity of the lipoxygenase and microperoxidase catalysts are retained. Examples of the use of this assay identify lipoxygenase inhibitors are shown in figures 3-5.
- the Assay according to the invention can be performed according to the scheme shown in figure 2.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
A method for identifying inhibitors of a lipoxygenase enzyme, the assay comprising: contacting a lipoxygenase enzyme with a test Compound, a lipoxygenase enzyme Substrate and oxygen; adding a f luorometric reagent and a peroxidase; measuring the fluorescent signal; determining the amount of enzyme inhibition by the test Compound.
Description
Lipoxygenase Enzyme Assay
BACKGROUND OF THE INVENTION
APPLICATION DATA
This application claims benefit to US provisional application serial no. 60/820,390 filed July 26, 2006.
1. TECHNICAL FIELD This invention relates to methods and kits for lipoxygenase enzymes which catalyze the oxygen-dependent oxidation of fatty acid substrates (linoleic acid and arachidonic acid are common examples) to form hydroperoxy-fatty acid products. The methods and kits are useful for detecting inhibitors of such enzymes.
2. BACKGROUND INFORMATION
Lipoxygenase enzymes catalyze the oxygen-dependent oxidation of fatty acid substrates (linoleic acid and arachidonic acid are common examples) to form hydroperoxy-fatty acid products. Enzymes have been purified from diverse organisms that display a broad range of substrate specificity and product specificity (i.e. the site of oxidiation within the fatty acid).
Several assay procedures have been published in the literature but each has particular limitations that make high-throughput screening difficult. The simplest assay is the spectrophotometric monitoring of the hydroperoxy-fatty acid product; the hydroperoxy- moiety absorbs light at 234 nm and can therefore be easily monitored with a spectrophotometer. As many potential inhibitors absorb light at this wavelength, this assay format is prone to interference from the very compounds we seek. Another method of assaying for lipoxygenase activity is to monitor the consumption of oxygen using a Clark electrode; this method is neither sensitive nor amenable to high- throughput. Another assay that has been used is to determine the concentration of hydroperoxy (or the chemically-reduced hydroxy-derivatives) fatty acids by separation from the substrate on a high-performance liquid chromatography (HPLC) system (for example, Yamamoto et al. (1990) Methods in Enzymology, 186, 371-380). These assays, while accurate, are heterogeneous and time consuming and are therefore not
amenable to screening large numbers of compounds. Two different colorimetric assay formats have been developed that utilize the oxidation state of the hydroperoxy product to couple product formation to color formation. Both assays are conducted in two steps and differ on the colorimetric reagent. After product has been formed, the color- forming reagent is added and the color is measured on a spectrophotometer. One assay used a xylenol orange :iron(II) complex (Waslidge et al. (1995) Anal. Biochemistry, 231, 354-358) and the second assay used hemoglobin as the catalyst) and N-benzoyl leucomethylene (Auerbach et al. (1992) Anal. Biochemistry, 201, 375-380) as the colorimetric reagent. These assays offer improved sensitivity over the direct spectrophotometric assay (~ 10-fold) and improved throughput when compared to the HPLC method. However, colorimetric assays suffer from a small signal-to-background window in which to measure a signal. Kratky et al. have published a very sensitive assay of lipoxygenases based upon chemiluminescent detection (Kratky et al. (1999) Biochimica et Biophyscia Acta, 1437, 13-22). The hydroperoxy-fatty acid product of lipoxygenase is reacted with isoluminol and microperoxidase to form an electronically excited form of 4-aminophthalate that emits a photon upon its decay. Because chemiluminescence is very short lived, each individual assay must be initiated and completed before proceeding to the next assay. This process makes the assay unsuitable for a high-throughput approach.
Molecular Probes (now part of Invitrogen) has published an assay for hydrogen peroxide detection that employs Amplex Red® (N-acetyl-3,7-dihydroxyphenoxazine) or Amplex UltraRed® and uses horseradish peroxidase as the redox catalyst instead of microperoxidase (Zhou et al. (1997) Anal. Biochemistry, 253, 162-168). While they sell many kits based upon the ability to couple hydrogen peroxide with Ample Red® oxidation, they do not mention the ability use the Amplex Red® reagent to detect hydroperoxy-fatty acids nor do any of their present reagents use microperoxidase as the redox catalyst.
The present inventors have designed an assay format to enable the identification of inhibitors of lipoxygenase enzymes. This assay represents a significant advantage over previous assay formats as the sensitivity and uniqueness of the signal render the format more amenable to high-throughput screening.
BRIEF SUMMARY OF THE INVENTION
It is therefore an object of the invention to provide a method to identify inhibitor of lipoxygenase enzymes.
It is a further object of the invention to provide a kit comprising an assay to identify inhibitor of lipoxygenase enzymes.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 : Schematic depiction of microperoxidase catalyzing a redox reaction between the hydroperoxy-fatty acid product and the Amplex UltraRed® to generate the highly fluorescent product resorufin. The amount of resorufin is then determined using fluorescence spectroscopy.
Figure 2: Schematic depiction of the fluorometric lipoxygenase assay that would be used to characterize the activity of a 15 -lipoxygenase.
Figures 3-5: Inhibition of x-lipoxygenase by representative compounds of varying potencies. The y-axis is Percent of Control and the x-axis units are in microM.
DETAILED DESCRIPTION OF THE INVENTION
To increase assay sensitivity and retain high-throughput features (homogenous assay that can be easily automated), the present invention provides a new assay for lipoxygenase which is an improvement from the historical assays described above. After the lipoxygenase has been incubated with the fatty acid substrate (linoleic acid or arachidonic acid) and oxygen, microperoxidase (a catalyst) and Amplex UltraRed® are added. The microperoxidase catalyzes a redox reaction between the hydroperoxy-fatty acid product and the Amplex UltraRed® to generate the highly fluorescent product resorufin. The amount of resorufin is then determined using fluorescence spectroscopy (excitation at 530 nm and emission at 580 nm). See figure 1. This assay improves the sensitivity ~ 10-fold over that observed in the colorimetric assays and generates a
fluorescent signal that is both stable and free from compound interference as very few compounds fluoresce in this range.
In the broadest generic embodiment, there is provided a method for identifying inhibitors of lipoxygenase enzymes, the assay comprising:
contacting a lipoxygenase enzyme with a test compound, a lipoxygenase enzyme substrate and oxygen; adding a fluorometric reagent and a peroxidase; measuring the fluorescent signal; determining the amount of enzyme inhibition by the test compound.
In second generic embodiment, there is provided a kit for determining the amount of lipoxygenase enzyme inhibition by a test compound comprising: a lipoxygenase enzyme; a lipoxygenase enzyme substrate; oxygen; a peroxidase and a fluorometric reagent.
The above kit can further contain a positive control that comprises a mock test compound. Said mock test compound having no or negligible lipoxygenase enzyme inhibition.
The Enzymes have been purified from diverse organisms that display a broad range of substrate specificity and product specificity. The assay as it is routinely performed is summarized in the scheme from the example section below but alterations apparent to those of ordinary skill in the art can be made. For instance, the incubation time or temperature can be adjusted but it is ideal to adjust them such that the enzyme activity is within the linear response range. The assay has been performed at various scales (cuvet, 96 or 384 well) and is expected to work at any scale required within any desired reaction vessel (e.g. polypropylene micro-plate or polystyrene cuvet). Any lipoxygenase enzyme that produces a hydroperoxy product, irrespective of stereo- specificity, is capable of being assayed by this technique, including 15 -lipoxygenase from humans or soybean, 12-lipoxygenase and 5 -lipoxygenase. Any substrate of the
lipoxygenase enzyme can be used; this could include, but is not limited to, free fatty acids or esterified fatty acids of varying composition (e.g. arachidonic acid, linoleyl- phosphatidyl choline, low-density lipoprotein, etc.). While Amplex UltraRed® is the preferred fluorometric reagent in this protocol, Amplex Red® or any reagent that results in the production of a fluorescent molecule with similar fluorescence (excitation maximum of 530 ± 25nm and emission maximum of 580 ± 25nm) can also be used. Similarly, microperoxidase may be substituted with any peroxidase that catalyzes the reaction between the hydroperoxide product and the fluorometric reagent (i.e. Amplex UltraRed® in the preferred embodiment). The solutions used for the lipoxygenase reaction and the microperoxidase reaction may also be modified from the specified conditions so long as activity of the lipoxygenase and microperoxidase catalysts are retained. Examples of the use of this assay identify lipoxygenase inhibitors are shown in figures 3-5.
EXAMPLES
The following examples are offered to illustrate, but not to limit the present invention.
The Assay according to the invention can be performed according to the scheme shown in figure 2.
All referenced cited in this application are incorporated herein by reference in their entirety.
Claims
1. A method for identifying inhibitors of a lipoxygenase enzyme, the assay comprising:
contacting a lipoxygenase enzyme with a test compound, a lipoxygenase enzyme substrate and oxygen; adding a fluorometric reagent and a peroxidase; measuring the fluorescent signal; determining the amount of enzyme inhibition by the test compound.
2. The method according to claim 1 wherein the lipoxygenase enzymes are chosen from 15 -lipoxygenase, 12-lipoxygenase and 5 -lipoxygenase.
3. The method according to claim 2 wherein the substrate of the lipoxygenase enzyme is a free fatty acid or esterifϊed fatty acid.
4. The method according to claim 3 wherein the fluorometric reagent is a reagent that results in the production of a fluorescent molecule with an excitation maximum of 530 ± 25nm and emission maximum of 580 ± 25nm.
5. The method according to claim 4 wherein
the fluorometric reagent is Amplex UltraRed® and the peroxidase is microperoxidase.
6. A kit for determining the amount of lipoxygenase enzyme inhibition by a test compound comprising: a lipoxygenase enzyme; a lipoxygenase enzyme substrate; oxygen; a peroxidase and a fluorometric reagent.
7. The kit according to claim 6 further comprising a positive control.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US12/373,540 US20090253158A1 (en) | 2006-07-26 | 2007-07-23 | Lipoxygenase Enzyme Assay |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US82039006P | 2006-07-26 | 2006-07-26 | |
| US60/820,390 | 2006-07-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2008014198A1 true WO2008014198A1 (en) | 2008-01-31 |
Family
ID=38610715
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US2007/074075 WO2008014198A1 (en) | 2006-07-26 | 2007-07-23 | Lipoxygenase enzyme assay |
Country Status (2)
| Country | Link |
|---|---|
| US (1) | US20090253158A1 (en) |
| WO (1) | WO2008014198A1 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2520006A1 (en) * | 1982-01-21 | 1983-07-22 | Toyo Jozo Kk | PROCESS FOR THE QUANTITATIVE MEASUREMENT OF UNSATURATED FATTY ACIDS |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ596024A (en) * | 2006-08-07 | 2013-07-26 | Ironwood Pharmaceuticals Inc | Indole compounds |
-
2007
- 2007-07-23 US US12/373,540 patent/US20090253158A1/en not_active Abandoned
- 2007-07-23 WO PCT/US2007/074075 patent/WO2008014198A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2520006A1 (en) * | 1982-01-21 | 1983-07-22 | Toyo Jozo Kk | PROCESS FOR THE QUANTITATIVE MEASUREMENT OF UNSATURATED FATTY ACIDS |
Non-Patent Citations (5)
| Title |
|---|
| ANTHON GORDON E ET AL: "Colorimetric method for the determination of lipoxygenase activity", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, vol. 49, no. 1, January 2001 (2001-01-01), pages 32 - 37, XP002456826, ISSN: 0021-8561 * |
| AUERBACH B J ET AL: "A SPECTROPHOTOMETRIC MICROTITER-BASED ASSAY FOR THE DETECTION OF HYDROPEROXY DERIVATIVES OF LINOLEIC ACID", ANALYTICAL BIOCHEMISTRY, vol. 201, no. 2, 1992, pages 375 - 380, XP009091677, ISSN: 0003-2697 * |
| CATHCART R ET AL: "DETECTION OF PICOMOLE LEVELS OF HYDRO PER OXIDES USING A FLUORESCENT DI CHLORO FLUORESCEIN ASSAY", ANALYTICAL BIOCHEMISTRY, vol. 134, no. 1, 1983, pages 111 - 116, XP009091584, ISSN: 0003-2697 * |
| KRATKY DAGMAR ET AL: "A sensitive chemiluminescence method to measure the lipoxygenase catalyzed oxygenation of complex substrates", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1437, no. 1, 29 January 1999 (1999-01-29), pages 13 - 22, XP004277168, ISSN: 0006-3002 * |
| ZHOU MINGJIE ET AL: "A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: Applications in detecting the activity of phagocyte NADPH oxidase and other oxidases", ANALYTICAL BIOCHEMISTRY, vol. 253, no. 2, 15 November 1997 (1997-11-15), pages 162 - 168, XP002456845, ISSN: 0003-2697 * |
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| Publication number | Publication date |
|---|---|
| US20090253158A1 (en) | 2009-10-08 |
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